This invention relates to imaging systems. In particular, the invention relates to imaging systems used for the acquisition, compilation, and analysis of fluorescent images.
The system described herein is used for the automated acquisition, compilation, and analysis of images of fluorescently labeled samples of different types (the xe2x80x9cInventionxe2x80x9d). Many areas of scientific investigation require the use of imaging equipment. For example, in the field of genomics research, imaging equipment is used to acquire, compile, and analyze the results from testing of deoxyribonucleic acid (xe2x80x9cDNAxe2x80x9d) xe2x80x9cmicroarraysxe2x80x9d (also know as xe2x80x9cgene chip arraysxe2x80x9d, xe2x80x9cbiochipsxe2x80x9d, and other designations).
Microarrays are prepared as a means to match known and unknown DNA samples based on hybridization principles, for example, to identify gene sequences or to determine gene expression levels. In one method, microarrays can be made by xe2x80x9cspottingxe2x80x9d collections of suspended, purified DNAs onto a substrate. In a typical production method, a microarray robot places drops of individual DNA types onto a substrate, such as a glass slide, in a grid design. The grid may contain thousands of DNA spots of different base pair sequences that are fixed to the substrate. cDNA xe2x80x9cprobesxe2x80x9d are then tested by hybridizing them to the prepared DNA microarray. If an individual cDNA probe is complementary to the sequence of DNA on a given spot, the cDNA will hybridize to the spot and the hybridization may be detected by its fluorescence. In this manner, each spot in the microarray may act to assay the presence of a different cDNA.
After the cDNA probes have hybridized to the microarray and any free probe has been removed, the microarray may be scanned to evaluate the comparative binding levels of individual probes. cDNA probes hybridized to DNA spots in the microarray may be detected through the use of different colored fluorophores or dyes that emit light at differential, characteristic wavelengths when excited by an illumination source. Microarray spots with more bound probe will fluoresce more intensely. The emitted light is captured by a detector, such as a charge-coupled device (CCD) or a photomultiplier tube (PMT), which records its intensity. The recorded data is stored or processed for further analysis. The detector for fluorescence emitted from the microarray is sensitive to the emission wavelength but filters out the excitation wavelength; in this way, the fluorescent emission of interest can be separated from the excitation light scattered off the substrate.
The use of robotically-prepared microarrays has increased the need for automated equipment to more quickly and accurately process and analyze test samples. Such automated equipment must be able to handle a high volume of samples with consistent and reproducible results. It is also desirable to have means to selectively filter emission and excitation wavelengths with reduced crosstalk between channels. It is also desirable to have reduced background in the scanned image, to be able to determine intensity thresholds, and to have more uniform detection efficiency across a scanned sample.
In contrast to existing systems that typically can accommodate only one test sample, the present Invention is fully automated and accommodates up to twenty-four samples on substrates, for example, on glass slides. The Invention""s CCD/arc lamp embodiment permits faster imaging than a stage-scanning instrument (such as General Scanning ScanArray 3000) because it uses full-field illumination and CCD detection. Depth of focus is deep enough to accommodate the added thickness of a glass cover slip on top of the sample, and allows for some variation in the height at which the sample is presented to the imaging system by the rotary stage. The Invention also permits the use of a large number of fluorophores at a lower cost than a laser-scanning instrument (such as Molecular Dynamics Avalanche and the GeneFocus DNAscope) because a new excitation band from the wide spectral band of the Invention""s arc lamp excitation source can be introduced through a simple filter change, in contrast to the introduction of an additional laser required in a laser-based system. The Invention also accommodates a straightforward extension of the dynamic range with a multiple exposure algorithm, which is much more difficult on a PMT-based system (General Scanning ScanArray 3000, Molecular Dynamics Avalanche, GeneFocus DNAscope) due to the non-linear nature of the PMT""s.
The Invention is a CCD and arc-lamp-based system used for the acquisition of fluorescent images, which can be optimized for imaging fluorescently labeled cDNA spotted on multiple glass slides. The Invention may also be applied in other uses, for example, to image other fluorescently labeled material, including proteins on other substrates, including gels and membranes, and for imaging histological or toxicology slides. With the excitation light blocked or turned off, the invention can also be used to image light emitting samples, such as chemiluminescently labeled materials, or radioisotopes in close proximity to a scintillating substrate.
The Invention uses a unique illumination and detection optical system that minimizes background from its high intensity arc-lamp illumination and thus allows detection of low-level fluorescence from the sample with a high-QE CCD detector.
The Invention incorporates a number of automated features, including automated presentation of multiple test samples to the viewing area by user-selected criteria; auto-exposure of test samples, which allows walk-away use of the instrument while imaging multiple samples; automated illumination-variation correction; and an automated technique for extending the dynamic range of the Invention past the bit depth of the CCD.
The optical system of the Invention allows for high-speed (approximately  less than 1 sec.) collection of images from bright samples with the CCD camera, and also allows longer integration times for collecting low-level fluorescence. Full-field illumination, in combination with integrating detection on the CCD, allows for low-fluorescence image collection, while minimizing fluorophore damage and bleaching, as compared to laser scanning systems. The optical system also allows for viewing a large area (approximately 30xc3x9740 mm) while eliminating sufficient background from the arc lamp to view low-level fluorescence. The arc lamp supplies a full spectrum of excitation light that can be adapted for different fluorophores by changing the waveband of the filters, whereas laser systems are limited to specific excitation lines. The linear behavior of the CCD detection allows for a straightforward method to increase the dynamic range of the image data by taking multiple exposures of the sample.